This proposal addresses RFA # RFA-GM-05-002 Protein Structure Initiative. The primary goal is structure determination of membrane proteins. Genes of membrane proteins that correspond to three or more membrane crossings will be clustered so that representatives can be selected from each cluster without known structure(s). The list will be dynamically re-prioritized as new structures emerge. Large-scale comparative modeling is to be automated and applied to proteins of known structure, a central component of structural genomics necessary to maximize coverage of sequence space. At each reprioritization, the next 30 highest priority genes will be cloned. The organisms selected are two bacterial, one archaebacterial species, and H.sapiens. The bacterial proteins are selected since they are most readily expressed in high yield and have so far yielded the highest density of membrane protein structures. H. sapiens is selected as the only eukaryote because human protein structures can instruct in structure-based drug design. These genes are to be expressed with C-terminal antibody affinity tags, for wild type and seleno methionylated proteins, sequentially through four expression systems, (i) in E. coli ii) by the novel Mistic system that allows for expression of eukaryotic proteins in E. coli, (iii) by the use of gene redesign to optimize codon usage for expression in E. coli, and (iv) by cell-free in vitro synthesis. Any human membrane proteins that are not successfully expressed in monodisperse form by these methods will be submitted to the NIH 'Roadmap Initiative'Center for Eukaryotic Membrane Protein Expression. Proteins will be purified, and characterized to assure structural homogeneity, monodispersity, correct mass using SELDI mass spectrometry, and correct folding. Proteins will be subject to three dimensional crystallization using an array of different detergents. Use of different lipids is to be evaluated for inclusion in this stage. Structures will be determined at the tunable ALS beamlines 8.3.1 and 5.02. Those that do not succeed in yielding adequate resolution will be subject to two dimensional crystallization in a lipid bilayer for two dimensional electron crystallography. Those that do not succeed will be subject to single particle electron image reconstruction if they are large, or to NMR structure determination if they are small (<20 kDa.). Results from successful structure determinations, and knowledge of clusters being attempted, including failures, will be instantly accessible to the public.